Selecting animals, such as boars, with high quality sperm is critical for livestock farming/industry. For example, pork is the most consumed meat in the world, over 100 million tons of pork meat is produced every year, and there are two billion living domestic pigs at any given time. The US and the EU constitute a third of the production, and almost all piglets born there are the result of artificial insemination (AI). AI has dramatically increased the genetic variability of these animals and enhanced the overall quality of the pork, especially the reduction of fat content that previously made it undesirable for many potential customers. It is critical that the boars, as well as other mammalian males, selected for breeding have high quality sperm, so that the success rate of fertilization is high and insemination results in frequent and healthy offspring.
Mammalian fertilization is initiated when sperm bind to the specialized extracellular matrix surrounding the egg, known as the zona pellucida (ZP). This binding triggers a signaling cascade that leads directly to acrosomal exocytosis (Yanagimachi, 1994). The induction of this reaction is indicated microscopically by the fusion of the plasma membrane with the outer membrane of the acrosome, a sperm organelle lying just beneath the plasma membrane. The fused membranes undergo vesiculation and fenestration, forming membrane blebs that slough off the sperm head, revealing the inner acrosomal membrane. Adhesion molecules on this membrane interact with the ZP, enabling the acrosome-reacted sperm to bind to and penetrate this matrix. The sperm enters the perivitelline space and fuses with the egg cell, thus completing the cellular binding events necessary for fertilization (Lopo et al., 1982; Yanagimachi, 1994).
Evidence that carbohydrate recognition plays a major role in mediating sperm-egg binding was initially obtained in several invertebrate marine species (Lopo et al., 1982; Mengerink and Vacquier, 2001; Monroy, 1965; Vacquier and Moy, 1977). Based on these findings, the natural assumption was that mammalian sperm-egg binding would also rely on lectin-like interactions. Defining the molecular basis underlying this binding event has been the subject of a major research effort ever since murine and porcine ZP (pZP) glycoproteins were initially isolated over three decades ago (Bleil and Wassarman, 1978; Dunbar et al., 1978). This interaction has also been investigated in the rat, hamster, and guinea pig (Tulsiani et al., 1997). Recently, the inventors' study also implicated the sialyl-Lewisx sequence expressed on the human ZP as a carbohydrate ligand that mediates human gamete binding (Pang et al., 2011).
Many studies indicate that a lectin-like egg binding protein (EBP) positioned on the mammalian sperm plasma membrane binds to glycans presented on the ZP during the initial stages of fertilization (Clark, 2010; Tulsiani et al., 1997). Evidence obtained in a somatic cell adhesion system also supports the presence of lectin-like EBPs on the surface of porcine and murine sperm (Clark, 2010; Sutton-Smith et al., 2007).
Classical lectins often display apparent Kd values in the nM range for glycoconjugates bearing their multivalent ligands (Dam et al., 2009). The possibility was considered that lectin-like EBPs require the appropriate presentation of carbohydrate ligands to mediate sperm binding, but do not require the simultaneous recognition of the ZP protein backbone. The glycans associated with mouse, pig and human ZP glycoproteins have now been sequenced (Clark, 2010; Hedrick, 2008 Noguchi and Nakano, 1993; Pang et at, 2011). Glycomic analyses of many glycoproteins unrelated to reproductive function have also been performed since 1970 (North et al, 2009). However, a direct testing of sperm fertility based on binding activity and abilities to undergo acrosomal exocytosis has never been considered or successfully attempted.
Therefore, there is a need to provide new testing methods and apparatus for determining fertility of a mammal with specific tests focusing on sperm motility, morphology, binding ability to an egg and ability for undergoing acrosomal exocytosis.